Isolation, Culture and Expansion of IFP-MSC
All experiments using human cells were performed in accordance with relevant guidelines and regulations. Human IFP-MSC were isolated from IFP tissue obtained from de-identified, non-arthritic patients that were age- and sex-matched (n=5; three males and two females; 36.6±12.9 years old) undergoing elective knee arthroscopy at the Lennar Foundation Medical Center at the University of Miami. Informed consent was obtained from all participants and/or their legal guardians. All procedures were carried out following approval by the University of Miami IRB not as human research (based on the nature of the samples as discarded tissue). IFP tissue (5-10 cc) was mechanically dissected and washed repeatedly with Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma Aldrich, St Louis, MO, USA), followed by enzymatic digestion using 235 U/ml Collagenase I (Worthington Industries, Columbus, OH, USA) diluted in DPBS and 1% bovine serum albumin (Sigma) for 2 hours at 37oC with agitation. Enzymatic digestion was inactivated with complete media with DMEM low glucose GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; VWR, Radnor, PA, USA), washed and seeded at a density of 1x106 cells/175 cm2 flask in chemically-reinforced (Ch-R) Mesenchymal Stem Cell Growth Medium 2 (PromoCell, Heidelberg, Germany). Forty-eight hours (48h) post-seeding, non-adherent cells were removed by DPBS gentle rinsing and fresh media were replenished accordingly.
All MSC were cultured at 37°C 5% (v/v) CO2 until 80% confluent as passage 0 (P0), then passaged at a 1:5 ratio until P3, detaching them with TrypLE™ Select Enzyme 1X (Gibco, ThermoFisher Scientific) and assessing cell viability with 0.4% (w/v) Trypan Blue (Invitrogen, ThermoFisher Scientific).
Immunophenotype
Flow cytometric analysis was performed on P3 IFP-MSC (n=3). Briefly, 2.0 x 105 cells were labelled with monoclonal antibodies specific for: CD44, CD73, CD90, CD105, CD107α (Biolegend, San Diego, CA), CD146 (Miltenyi Biotec, Auburn, CA), CD166, NG2, HLA-DR (BD Biosciences, San Jose, CA), and the corresponding isotype controls. All samples included a Ghost Red Viability Dye (Tonbo Biosciences, San Diego, CA). All samples included a Ghost Red Viability Dye (Tonbo Biosciences, San Diego, CA, USA). Data (20.000 events collected) were acquired using a Cytoflex S (Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza analysis software (Beckman Coulter).
Isolation and validation of IFP-MSC derived exosomes
IFP-MSC derived exosomes (IFP-MSC EXOs) were isolated from IFP-MSC conditioned medium by a step-wise ultracentrifugation method and CD63-immunomagnetic purification. Briefly, conditioned media from IFP-MSC groups cultured in exosome-depleted Ch-R medium56 are filtered through a 0.22µm filter to remove debris and large vesicles, and differentially centrifuged for 2,000xg for 10 min, 10,000xg for 30 min, and ultracentrifuged for 120,000xg for 16hr57. Pre-enriched exosome preparations were incubated with the Dynabeads®-based Exosome-Human CD63 Isolation/Detection Reagent (Invitrogen) and using a magnetic separator, exosome preparations are further purified. Samples from each group were assessed for biophysical and biochemical characterization58.
Quantity and size determination were performed by nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern). Samples were diluted in PBS from 1:10 to 1:100 depending on the initial sample concentration. The software settings for analysis were: detection threshold 5; room temperature; number of frames 30 and measurement time 30 s. The size distribution and particle concentration each represent the mean of five individual measurements.
Exosomes presence was validated by CD9 (Invitrogen) expression in CD63+-gated particles by flow cytometry. Data (20.000 events collected) were acquired using a Cytoflex S (Beckman Coulter) and analyzed using Kaluza analysis software (Beckman Coulter).
The functional assessment of IFP-MSC EXOs was performed in two concentrations corresponding to exosomes secreted from 5 x 105 (EXO) and 5 x 106 (10x EXO) IFP-MSC. For IFP-MSC EXOs tracking, exosomes were stained with PKH26 red fluorescent membrane staining kit (Fluorescent Cell Linker Kits, Sigma) according to manufacturer’s instructions and co-cultured with target cells in functional assessments.
miRNA profile of IFP-MSC EXOs
miRNA and protein were extracted from IFP-EXOs using Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Total exosome miRNA (1μg) was used for first-strand cDNA synthesis with All-in-One miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD).
Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia) were performed using 1000 ng cDNA per IFP-MSC sample (n=2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC). Data analysis was performed using qPCR result with GeneCopoeia’s online Data Analysis System (http://www.genecopoeia.com/product/qpcr/analyse/). Mean values were normalized to small nucleolar RNA, C/D box 48 (SNORD48), expression levels were calculated using the 2−ΔCt method. Putative miRNA interactomes were generated using a miRNet centric network visual analytics platform (https://www.mirnet.ca/). The miRNA target gene data were collected from well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with 34 cycles cut-off point) were represented in a topology miRNA-gene interactome network using force atlas layout and hypergeometric test algorithm. InfoMap algorithm was also used to identify miRNAs with higher node degree that act as hubs in a gene network.
Protein profile of IFP-MSC EXOs
Multiplex protein arrays of 35 cytokines and 41 growth factors (RayBio® C-Series, RayBiotech, Peachtree Corners, GA) were used to determine IFP-MSC EXOs protein cargo (n=2). One mL of IFP-MSC EXOs protein extract was used for each assay following the manufacturer’s instructions. Data shown represent 40 sec exposure in FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA). Results were generated by quantifying the mean spot pixel density of each array using protein array analyser plugin using ImageJ software (Fiji/ImageJ, NIH website). The signal intensities were normalized with the background whereas separate signal intensity results represent the average pixel density of two spots per protein. Signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample.
Pathway analysis
Putative interactomes were generated by Search Tool for Retrieval of Interacting Genes/Proteins (STRING 11.0; available from: http://string-db.org) database using interaction data from experiments, databases, neighbourhood in genome, gene fusions, co-occurrence across genomes, co-expression and text-mining. An interaction confidence score of 0.4 was imposed to ensure high interaction probability. K-means clustering algorithm was used to organize proteins into 3 separate clusters per condition tested, discriminated by colours. Venn diagrams were used to demonstrate all possible relations between naïve and/or TIC-primed IFP-MSC cultured under all three different conditions for the significantly (p<0.05) altered proteins. Functional enrichments related to biological process, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and reactome pathways were presented in radar graphs for proteins tested.
Synoviocyte inflammation assay
Passage 1 synoviocytes (SYN) expanded in synoviocyte medium (ScienCell, Carlsbad, CA) were subsequently primed with TIC inflammatory/fibrotic cocktail (15 ng/ml TNFα, 10 ng/ml IFNγ, 10 ng/ml CTGF) for 72h. On day 3, synoviocyte/exosome (SYN/ IFP-MSC EXOs) co-cultures were performed using EXO and 10x EXO for each sample. Co-cultures were fed with synoviocyte medium or synoviocyte medium + TIC inflammatory/fibrotic cocktail for 72 hours according to the initial non-induced or TIC-primed cohorts designation.
CCK-8 cytotoxicity assay (Cell Counting Kit-8, Sigma) was performed in non-induced or TIC-primed SYN and SYN/ IFP-MSC EXOs according to manufacturer’s instructions. EXO and 10x EXO cytotoxicity was determined by measuring optical densities of individual wells at 450nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA).
RNA extraction from SYN cultures was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD) according to manufacturer’s instructions. Total RNA (1µg) was used for reverse transcription with SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). A pre-designed 28 gene Taqman low density cytokine array (TLDA, Applied Biosystems) was performed using 1000 ng cDNA per culture and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC). Data analysis was performed using DataAssist software v2.0 (Applied Biosystems, LLC). Mean values were normalized to GAPDH, expression levels were calculated using the 2−ΔCt method. Values were represented in a fold change heatmap as the relative fold change of the TIC SYN/EXO or TIC SYN/10x EXO to TIC SYN (reference sample, 2−ΔCt= X sample/X reference sample).
Secretory profiles of non-induced and TIC-primed cohorts were evaluated using multiplex protein arrays of 40 cytokines (RayBio® C-Series, RayBiotech). One mL of conditioned media was used for each assay following the manufacturer’s instructions. Data shown represent 40 sec exposure in FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA). Results were generated by quantifying the mean spot pixel density of each array using protein array analyser plugin using ImageJ software (Fiji/ImageJ, NIH website). Signal intensities were normalized with the background whereas separate signal intensity results represent the average pixel density of two spots per protein. The signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample.
Macrophage polarization assay
Human monocytes (THP-1, ATCC) were differentiated into macrophages using PMA/IO (Phorbol 12-myristate 13-acetate/Ionomycin) and polarized to M1 macrophages by M1-macrophage generation medium (PromoCell). PMA/IO-stimulated THP-1/IFP-MSC EXOs co-cultures were performed for 2 days using EXO and 10x EXO. Generated macrophages inflammation-related protein profiling was performed by multiplex protein arrays whereas their polarization status was assessed using macrophage polarization qPCR array (ScienCell).
A CCK-8 cytotoxicity assay (Cell Counting Kit-8, Sigma) was performed in non-induced or PMA/IO-primed THP-1 and THP-1/IFP-MSC EXOs according to manufacturer’s instructions. EXO and 10x EXO cytotoxicity was determined by measuring optical densities of individual wells at 450nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA).
Secretory profiles of PMA/IO-primed cohorts were evaluated using multiplex protein arrays of 40 cytokines (RayBio® C-Series, RayBiotech). One mL of conditioned media was used for each assay following the manufacturer’s instructions. Data shown represent 40 sec exposure in FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA). Results were generated by quantifying the mean spot pixel density of each array using protein array analyser plugin using ImageJ software (Fiji/ImageJ, NIH website). The signal intensities were normalized with the background whereas separate signal intensity results represent the average pixel density of two spots per protein. The signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample.
RNA extraction from THP-1 cultures was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD) according to manufacturer’s instructions. Total RNA (1µg) was used for reverse transcription with SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). A pre-designed 40 gene Human Macrophage Polarization array (GeneQuery™ Human Macrophage Polarization Markers qPCR Array Kit, ScienCell) was performed using 1000 ng cDNA per culture and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC). Mean values were normalized to GAPDH, expression levels were calculated using the 2−ΔCt method. Values were represented in a stacked bar plot for M0, M1, and M2 polarization as the relative fold change of the PMA/IO + THP-1/EXO or PMA/IO + THP-1/10xEXO to PMA/IO + THP-1 (reference sample, 2 − ΔCt= X sample/X reference sample).
Mono-iodoacetate model of acute synovial/IFP inflammation
All animal experiments were performed in accordance with relevant guidelines and regulations. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami, USA (approval no. 21-030 LF) and conducted in accordance to the ARRIVE guidelines59. Twelve (#12) 10-week old male Sprague Dawley rats (mean weight 250 g) were used. The animals were housed to acclimate for 1 week before the experiment initiation. One rat was housed per cage in a sanitary, ventilated room with controlled temperature, humidity, and under a 12/12 hour light/dark cycle with food and water provided ad libitum.
Acute synovial/IFP inflammation was generated by intra-articular injection of 1 mg of mono-iodoacetate (MIA) in 50 µl of saline into the right knee. Briefly, under isoflurane inhalation anesthesia, rat knees were flexed 90º and MIA was injected into the medial side of the joint with a 27G needle using the patellar ligament and articular line as anatomical references. Four (4) days later, a single intra-articular injection of EXO or 10x EXO in 50 µl of Euro-Collins solution (MediaTech) was performed (similar injection technique), having as control: 1) rat knees receiving MIA but not IFP-EXOs (Only MIA group); and 2) healthy rat knees. Animals were sacrificed at day 4 after IFP-EXOs injection (d8 in total). This short exposure to MIA has been shown to induce inflammatory changes within the synovium and adjacent IFP52.
Cytochemical staining and CD86/CD206 immunolocalization in situ
Rat knee joints were harvested by cutting the femur and tibia/fibula 1 cm above and below the joint line, muscles were dissected and removed, and joints were fixed with 10% neutral buffered formalin (Sigma-Aldrich) for 14 days at room temperature. Knee joints were decalcified, cut along the sagittal plane in half, embedded in paraffin, and serial 4 µm sections obtained. Hematoxylin and Eosin (H&E) staining was performed to evaluate the gross structure and morphology of the knee joints. Masson’s trichrome staining for collagen-rich fibrotic areas was used to evaluate for extent of fibrosis within the fat pad tissue. Microscope images of cytochemically stained tissues were acquired using × 10 objective Leica DMi8 microscope with Leica X software (Leica). Based on histochemical stainings, tissue synovitis/fibrosis was evaluated in 3 rat knees per condition and 4 microscopy fields per knee with Fiji/ImageJ software.
For anti-substance CD86 and CD206 immunofluorescence staining, sections were incubated with 1x citrate buffer solution at 60°C overnight for antigen retrieval, permeabilised with 1x PBS + 0.2% Triton X-100 for 20 minutes at room temperature, and incubated with blocking buffer (1x PBS + 0.1% Triton X-100 with 10% rabbit serum) for 1 hour at room temperature. In between different treatments sections were washed with 1x PBS. Mouse anti-rat CD86 monoclonal antibody and rabbit anti-rat CD206 polyclonal antibody (both from Abcam) were prepared in blocking buffer (1:500) and sections were incubated at 4°C overnight. Sections were washed with 1x PBS + 0.01% Triton X-100 and incubated for 2 hours with secondary antibodies containing Alexa Fluor488 conjugated goat anti-mouse and Alexa Fluor647 conjugated goat anti-rabbit IgG antibodies (Thermo Fisher Scientific) at room temperature. Controls were incubated with secondary antibodies only. All sections were rinsed with 1x PBS, mounted in prolong gold antifade reagent with DAPI (Invitrogen), and microscope images were acquired using Leica DMi8 microscope with Leica X software (Leica).
Statistical analysis
Normal distribution of values was assessed by the Kolmogorov-Smirnov normality test. Statistical analysis was performed using paired and unpaired Student’s t-test for normally distributed data and Wilcoxon (for paired data) or Mann Whitney (for unpaired data) test. In the presence of a non-normal distribution of the data, a one-way ANOVA was used for multiple comparisons. All tests were performed with GraphPad Prism v7.03 (GraphPad Software, San Diego, CA). Level of significance was set at p < 0.05.